1. Field of the Invention
This invention relates generally to techniques used in the promotion of wound healing. In particular, compositions of matter that promote the healing of wounds, methods of manufacture of wound healing promoting compositions, and methods of treatment that promote wound healing are encompassed within the scope of the present invention.
2. Description of the Related Art
Animals, including human beings are susceptible to a barrage of normal cuts and scrapes, as well as to much more serious wounds that may require medical attention.
Wounds may be the result of accidents or surgery. For the most part, such wounds heal at a fairly steady and slow rate, being affected by many factors including the nature and site of the wound and the physiological state of the animal.
The process of wound healing involves many complicated components. Immediately upon the injury insult, defense mechanisms inherent in normal body tissues are activated to restore continuity and tensile strength. Wound healing then occurs in three distinct phases.
First, is the phase of acute inflammatory response. Body fluids containing plasma proteins, fibrin, antibodies and various blood cells flow into the wound. Scab formation takes place and inflammation occurs within a few hours. Also, at this stage, neutrophils, monocytemacrophages come into play. During this acute phase, the wound is solely dependent on the closure material contained in the scab for strength.
Second, is the phase of fibroplasia. Here, via various enzymatic mechanisms, fibrin synthesis and accumulation takes place. This causes an increase in wound tensile strength and stimulation of fibroblast proliferation and growth. Fibroblasts secrete collagen, a fibrous protein as part of connective tissue. Collagen deposition begins from the fifth day and results in rapid gain in tensile strength of the wound.
The third phase is the maturational process. Tensile strength continues to increase from the cross-linking of collagen fibers. Deposition of fibrous connective tissue causes scar formation.
Collagen production is vital for the wound healing process. Collagen is the most prevalent protein in animals. It is an obligatory constituent of connective tissues and extra cellular matrices. Collagen networks in the tissues are responsible for establishing and maintaining the physical integrity of diverse extra cellular structures. Collagen, at molecular level, is defined as a protein comprised of lengthy domains of triple-helical confirmation. Collagenous scaffolding of extra cellular matrix includes genetically distinct types of collagen. During the normal wound repair, collagen neosynthesis and deposition of type III collagen is demonstrated in the earliest phase, i.e. 24 hr to 48 hr, period. From that point, a significant increase in type I collagen is associated with the mature wound fibroblasts and subsequent healing events. Because of its important role in the wound healing process, collagen production is a measure of the rate and quality of wound healing. As such, assays that measure collagen production are useful in experimental models to study wound healing.
The healing process is very much organ and tissue-type dependent. For example, intestinal tissue is physiologically a rapidly self emphasizing tissue and unlike other organs in that it must constantly be repaired. Intestinal repair is an ongoing process necessary to maintain normal function of the intestines. There is an almost constant need for repair in the intestines, where injury arises from aberrations in the digestive process or from ingested foods. In contrast to intestinal repair, the xe2x80x9cwound healingxe2x80x9d discussed in this application is caused by external factors of trauma and injury. Such sudden and external trauma injury requires intact and able host defense mechanisms.
The process of wound healing involves a complex system of local and remote (systemic) energy and substrate requirements and uses. For example, amino acids and sugars are needed as substrates for collagen and proteoglycan synthesis. Migration of fibroblasts and epithelial/endothelial cells during the wound healing process places additional systemic demands on the animal during the wound healing process. Wounded tissues have unique nutritional needs and physiological features. Lymphocyte participation in wound healing has been demonstrated (Peterson et al. (1987)). Alteration in the hosts T-cell dependent immune response has also been shown to influence wound healing. Cyclosporine and anti T-cell antibodies, both of which interfere with T-cell function, abrogate wound healing. Similarly, macrophages and their products are also involved in wound healing. Increased circulation usually results in rapid delivery of monocytes and PMN""s to the wound site. This in turn results in the elimination of bacterial contamination of the wound due to nonspecific killing mechanisms and also enhances the rate of wound healing. These various cell types are synthesized by the bone marrow.
In many cases, the wound healing process proceeds very slowly, particularly in animals having limited energy stores or diets low in energy substrate sources.
Purine and pyrimidine nucleotides are involved in almost all cellular processes and play a major role in structural, metabolic, energetic and regulatory functions. They make up the monomeric units of RNA and DNA; RNA synthesis is required for protein synthesis and DNA synthesis is required for growth and cell division. Adenosine triphosphate, an adenine nucleotide is the major source of chemical energy used in metabolism, driving almost all cellular processes. Nucleotides are physiological mediators in a number of metabolic processes. Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) regulate a large number of cellular events, and adenosine is important in regulating blood blow and smooth-muscle activity. Guanosine triphosphate (GTP) is involved in signal transduction, RNA structure, and microtubule formation. Many other nucleotides are involved in regulating other cellular processes. Nucleotides function as activated intermediates in the synthesis of glycogen and glycoproteins; they are also intermediates in the synthesis of phospholipids, and serve as methyl and sulfate donors. They are structural components in a number of coenzyme that are crucial in many metabolic pathways, and they function as allosteric effectors that control the regulatory steps of major metabolic pathways.
Nucleotides consist of a nitrogenous base (either a purine or a pyrimidine), a sugar, and one or more phosphate groups. The term nucleotide in the context of the title refers to the multiple forms in which purines and pyrimidines are found and does not imply a specific form of the compounds but all forms that contain purine and pyrimidine bases.
The major purine bases are adenine, guanine, hypoxanthine and xanthine. Uric acid is also found in significant levels. The major pyrimidine bases are uracil, thymine, and cytosine. Other pyrimidines and purines are also present in smaller amounts and they have significant roles particularly in RNA structure and function.
The nucleotides are phosphoric acid esters of nucleosides in which the phosphoric acid is esterified to one of the free pentose hydroxyl groups. Nucleotides occur in free form in significant amounts in a variety of cell types. They are also formed on partial hydrolysis of nucleic acids, particularly by the action of a class of enzymes called nucleases. Nucleotides containing 2-deoxy-D-ribose are deoxyribonucleotides; those containing D-ribose are ribonucleotides. A nucleoside, which does not have a phosphate group, is formed from a base and a pentose via a glycosidic bond between the N-1 nitrogen of a pyrimidine or the N-8 of a purine and the C-1xe2x80x2 carbon of the pentose. The pentose is ribose or 2xe2x80x2-deoxyribose. The major function of the 2xe2x80x2-deoxyribose nucleotides is in DNA. The ribonucleotides are the monomeric units of RNA but also serve in most other cellular and metabolic functions of nucleotides. The phosphoryl group of nucleotides is most commonly esterified to the C-1xe2x80x2 hydroxyl of the pentose. In cyclic nucleotides the phosphate is esterified to both the C-5xe2x80x2 and C-3 hydroxyl groups. The number of phosphate groups attached is indicated by a mono-, di- or tri-designation. In the discussion and description of the claims the term nucleotide will be used generally to mean a source of preformed purines and/or pyrimidines in various forms including RNA as well as individual purines and/or pyrimidines as bases, nucleosides or nucleotides. It does not generally (except as noted in specific examples) imply that one form is required.
Since there are two or more free hydroxyl groups in nucleosides, the phosphate group of nucleotides can potentially occur in more than one position on the sugar ring. In the case of deoxyribonucleotides, there are only two possible positions in 2-deoxyribose that can be esterified with phosphoric acid, namely, the 3xe2x80x2 and 5xe2x80x2 positions. Both 3xe2x80x2- and 5xe2x80x2-deoxyribonucleotides occur biologically. In the case of ribonucleotides, the phosphate group may be at the 2xe2x80x2, 3xe2x80x2, or 5xe2x80x2 position; all 3 types of ribonucleotides have been found as hydrolysis products of RNA, depending on conditions. Cyclic monophosphates of adenosine are also possible. However, the nucleotides that occur in the free form in cells are predominantly those having the phosphate group in the 5xe2x80x2 position, since the enzymatic reactions normally involved in nucleic acid synthesis and breakdown in cells proceed via nucleoside 5xe2x80x2-phosphates intermediates. Table 1 gives the nomenclature of the major ribonucleoside 5xe2x80x2-monophosphates (also called 5xe2x80x2-ribonucleotides) and deoxyribonucleoside 5xe2x80x2-monophosphates (also called 5xe2x80x2-deoxyribonucleotides). All the common ribonucleosides and 2xe2x80x2-deoxyribonucleosides also occur in cells as the 5xe2x80x2-diphosphates and the 5xe2x80x2-triphosphates, i.e., the 5xe2x80x2-pyrophosphoric and the 5xe2x80x2-triphosphoric acid esters of the nucleosides.
Purines and pyrimidines can be formed by de novo biosynthesis or salvage of preformed bases and interconversion to the desired compound. Almost all of the atoms in both bases are derived directly or indirectly from amino acids. Phosphoribosylpyrophosphate (PRPP) serves as the pentose source for both purine and pyrimidine biosynthesis and for salvage of bases. PRPP is formed from ribose-5-phosphate. Deoxyribonucleotides are subsequently formed from the ribonucleotides.
The pathway for purine biosynthesis consists of ten steps. The initial step involving PRPP and glutamine condensation catalyzed by PRPP aminotransferase is likely the rate limiting step and is feed-back inhibited by AMP and GMP. IMP is the first purine formed and it is converted to either AMP or GMP depending on cell needs. Regulation occurs at these steps also. The monophosphates of both purines and pyrimidines are readily converted to di- and triphosphates by various kinase enzymes using ATP as a phosphate source.
In pyrimidine biosynthesis, PRPP is not added until the intact pyrimidine is formed as orotic acid. OMP (orotidine-5xe2x80x2-monophosphate) is the first pyrimidine formed but it functions in the cell only as a precursor of other pyrimidines. UMP is formed from OMP and then CTP and TTP are derived from UMP. In eukaryotes regulation of pyrimidine synthesis occurs primarily at carbamoyl phosphate synthesis with inhibition by pyrimidine nucleotides and activation by purine nucleotides.
Deoxyribonucleotide synthesis is catalyzed by ribonucleotide reductase, an enzyme that converts both purine and pyrimidines to their deoxyribose forms. The reductase is controlled in a complex manner by both substrates and product to allow synthesis of equimolar levels of the various deoxyribonucleotides. Since the deoxynucleotides are used only for DNA synthesis the levels of the purine and pyrimidine need to be equal. Thymidine triphosphate is then formed as the monophosphate from deoxy-UMP. The levels of the deoxyribonucleotides are typically in the range of 2-60 mM while ribonucleotides are typically much higher with ATP concentration in the range of 2-10 mM and other ribonucleotides from 0.05-2 mM. Di- and monophosphates are typically lower than the triphosphates. Levels of both ribo- and deoxyribonucleotides will vary considerably depending on the phase of the cell cycle and under various metabolic conditions.
In primates uric acid is the end product of purine catabolism while other species can convert it to more soluble forms. The end products of pyrimidine catabolism are xcex2-alanine and xcex2-amino isobutyrate which are both soluble and easily excreted. Less is known about pyrimidine catabolism since no clinical effects of the end products occur. The catabolic pathways operate in the digestive system converting DNA and RNA and free nucleotides to nucleosides and free bases. Pyrimidine bases and nucleosides are taken up and readily incorporated into tissues. Dietary nucleotides appear to be important in support of cellular metabolism particularly in rapidly dividing tissues such as lymphoid cells and the intestine.
The uptake of purines and pyrimidines from the intestine and cellular turnover of nucleotides particularly from mRNA provides preformed bases that avoid the metabolic cost of de novo biosynthesis. Synthesis of both purines and pyrimidine consumes a significant amount of energy. It is important to note that role of amino acids in nucleotide synthesis and the salvage of dietary and cellular sources of nucleotides. A balance exists between these different pathways affording proper levels of nucleotides in cells with minimal metabolic expense.
The usefulness of dietary nucleotides in certain medical contexts is documented. The instant inventors and others have described the potential role of dietary nucleotides in several contexts. For example, dietary nucleotides are required for maintenance and recovery of host immune response (Van Buren et al. (1983) and Rudolph et al. (1990)). It has also been shown that there is increased activity of Lyt1+, IL2-R+ and Mac1+ cells in the tissues responding to alloimmune challenge (Van Buren et al. (1985) and Kulkarni et al. (1988)). Nucleotide supplementation has also been shown to provide an increase in both immunohemopoiesis (Kulkarni et al. (1992)) and resistance to infectious microorganisms (Kulkarni et al. (1986)). Nucleotide supplementation has also been described as reversing immunosuppression induced by protein starvation. (Pizzini et al. (1990)).
Several research groups have published works concerning the relationship of nucleotides to immune system functioning. Van Buren et al. (1985) relates to the role of dietary nucleotides in the processes of recognition of and sensitivity to foreign antigens and in lymphocyte proliferation to alloantigen or lectin stimulation. The present inventors have also described the importance of dietary sources of pyrimidines and purines, such as those in nucleic acids, in immune function and on gastrointestinal function. (Rudolph et al. (1990)) Normal cellular immune response has therefore been postulated to require a source of preformed nucleotides. The authors conclude that dietary sources of nucleotides are important to support optimal growth and function of metabolically active cells such as lymphocytes, macrophages and intestinal cells.
The role of dietary nucleotides in the immune response is further examined in Pizzini et al. (1990). In this series of studies, nucleotide restriction was tested using both a starvation malnutrition and a protein malnutrition in vivo model. Animals in the starvation malnutrition study receiving a diet supplemented with yeast RNA prior to the period of starvation (5 days) reportedly demonstrated an increase in spontaneous concanavalin A and phytohemagglutinin-stimulated blastogenesis in in vitro assays. In protein malnutrition studies, the return to any of the examined diets (chow diet, nucleotide-free diet, or nucleotide free diet supplemented with 0.25% yeast RNA) reportedly resulted in restoration of body weight, while only the RNA-supplemented and chow diets restored popliteal lymph node immune reactivity.
The usefulness of nucleotides in the repair or regeneration of intestinal gut cells in infants was the basis of the U.S. Pat. No. 4,994,442. This patent relates to a milk and non-milk based infant formula that includes nucleosides and/or nucleotides. As previously stated, this process of intestinal repair is continual and physiologically distinct from wound healing in response to trauma or insult, which is the goal of the present invention.
The role of dietary nucleotides in preventing the onset of infection has also been studied. In Kulkami et al. (1986), the present inventors present data relating to the role of dietary nucleotides (for example, dietary adenine, uracil or RNA) in maintaining animal resistance to Staphylococcus aureus. Fanslow et al. (1988) examines the relationship between dietary nucleotides and animal susceptibility to candidiasis. Studt et al. (U.S. Pat. No. 4,486,439) relates to a method for treating coccidial infections employing a formulation that includes, among other ingredients, 2-pyrimidine, 4-pyrimidine, 5-pyrimidine, 6-pyrimidine, 2-purine, 6-purine, 8-purine or 9-purine.
Dietary nucleotides have been implicated as having a role in relation to delayed cutaneous hypersensitivity (Kulkami et al. 1987) and in the fatty acid composition of erythrocyte membrane lipids in infants (DeLucchi et al. 1987).
Gil et al. (U.S. Pat. No. 5,066,500) relates primarily to a non-milk based infant formula that includes amino acids and is enriched with nucleotides and/or nucleosides (at least one of uridine, uridine-phosphate, guanosine or guanosine phosphate, adenosine or adenosine phosphate, cytidine or cytidine phosphate, inosine or inosine phosphate, or mixtures thereof). Examples are also provided of defined composition dietary supplements for adults suffering from such non-trauma or insult problems as energy-protein malnutrition, hypercatabolism, malabsorption-malnutrition syndromes, severe homeopathy, or chronic hematopathy. However, these formulations are not used to stimulate the immune system. Gut intestinal cell turnover is a normal, physiologic process, in which the inflammatory response plays no role. Healing of a traumatic wound, on the other hand, requires an inflammatory response as a necessary first step in wound healing.
A respiratory enzyme booster tablet that includes a combination of diphosphopyridine nucleotide, nicotinamide, adenosine-5-monophosphate and a carrier has been described in the Case patent (U.S. Pat. No. 4,308,257). The compound functions as a co-enzyme that acts in the cellular respiration process. An injectable treatment that includes diphosphopyridine nucleotide is also described. The use of a nucleotide compound in the absence of other ingredients however, has not been described, nor suggested as a potentially useful therapeutic agent. Also, these tablets are specifically designed to increase the rate of cellular respiration, a phenomenon that occurs in all cells. These formulations do not appear to play a role in the enhancement of collagen formation or wound-healing.
The Guari patent (EP No. 85,084, 1983) relates to a wound-healing and antiviral preparation which includes a dialysis concentrate of deproteinated calf s blood and a member of a very specific class of furanosylated, uracil derived compounds. These ingredients reportedly act xe2x80x9csynergisticallyxe2x80x9d to provide the described physiological effects.
The idea and process of nutritional therapeutic approach would have no side effects (toxic or untoward) as shown many times by the pharmacologic or chemotherapeutic interventions. Injury or trauma induced stress causes sudden loss of body fluids and nutrients, proper nutritional repletion can improve these losses. The effects may be sustaining and long term rather than symptomatic quick-fix afforded by other means. Nutritional modulation may help and improve the endogenous physiologic process in order to combat the wound-related trauma.
In reviewing the known related art, it becomes apparent that there has been no suggestion of the usefulness of nucleotides as pharmacologically active agents in the relatively complex, processes of wound healing. For example, the specific events important in wound healing of collagen formation, fibroblast proliferation and restoration and maintenance of host immune response have not been described or suggested to be enhanced through dietary supplementation with nucleotides.
Normal wound healing can be impaired by chronic infection, protein malnutrition, poor blood supply, vitamin deficiencies, previous radiation exposure, diabetes mellitus, corticosteroid therapy and deficiencies in the components of the host wound response. Obviously, many of these conditions are more likely to cause problems the longer a wound takes to heal. Additionally, escalating health care costs indicate a need for methods that promote wound healing. Therefore, any procedures that would aid in wound healing would be welcomed in the medical field.
The problems associated with wound healing are in part remedied by the compositions and methods of the present invention. The inventors have found that wound healing can be greatly enhanced by the inclusion of nucleotides and/or substances that include essential nucleotides, such as RNA, DNA, oligonucleotides, purine and pyrimidine bases, or any other source in a pharmaceutical preparation. Dietary nucleotides are also proposed by the present inventors to be useful in pretreatment regimens to enhance wound healing in, for example, surgery patients. The invention provides for the use of nucleotides in concentrations effective to promote wound healing. Great utility is realized with the described compositions and methods in enhancing the rate of wound healing, and the wound healing process in general. A more rapid wound healing process also is anticipated to reduce recovery time. Concomitant benefits would also include a reduction in medical costs and treatment, time away from work, and the incidence and severity of infection.
The inventors have shown that dietary nucleotides modulate various host immune parameters, especially in protein-malnutrition induced stress, nucleotide supplemented diets improve rapidly the host immune system. This has been shown by various in vivo assays examining the immunologic capacity as well, as evidenced by an increased resistance to sepsis observed by the present inventors. It is felt that utilization of exogenously supplied nucleotides by T-lymphocytes and macrophages of the body""s immune system is independent of provision of dietary protein. A unique quality of dietary nucleotides heretofore undescribed for any other nutritional substrate improves systemic host immune response, both specific and nonspecific, is therefore provided by the present invention. Such a boost of the immune response then, in turn, responds to the body""s requirements for alleviating insults. Such insults would include trauma, injury, either external or internal that would require immediate repair in order to maintain proper body physiology and function. The wound models described in this application examine such cases of injury.
The present invention contemplates a therapeutic agent for the promotion of wound healing. In one preferred embodiment, the therapeutic agent comprises a therapeutically effective concentration of nucleotides (i.e., effective to promote wound healing) in a pharmacologically acceptable carrier. The nucleotides contained in the xe2x80x9cactive compoundxe2x80x9d of the therapeutic agent may comprise RNA, adenine, uridine, any of the compounds contained in Table 1, or a combination thereof. In some preferred embodiments of this invention, the nucleotide component comprises RNA, adenine, uridine, inosine or a mixture thereof. While almost any level of nucleotide administration is expected to be of benefit in the wound healing process, it is anticipated that concentrations of about 0.10% to 0.50% (ranging from 0.00034 g/kg body wt/day to 0.17 g/kg body wt/day) will be particularly useful. These concentrations are for purines and pyrimidines in the form of nucleotides in the pure chemical sense, i.e. with a phosphate group. If nucleosides are administered, the concentrations will range from 0.00022 g/kg/day-0.12 g/kg/day, since nucleotides do not contain the weight of a phosphate group. As mentioned previously, the use of the term xe2x80x9cnucleotidexe2x80x9d elsewhere in the application means both nucleotide and nucleoside forms of the purines and pyrimidines. In the claims, a claim to concentrations of nucleotides, (i.e. 0.00034-0.17 g/kg/day) encompasses the equivalent amount of nucleoside (i.e. 0.00022-0.12 g/kg/day). A concentration of about 0.25% represents a most preferred embodiment of the present invention.
A decided practical advantage is that the nucleotides that comprise the active compounds of the present invention may be administered as a dietary supplement in any convenient manner, such as by the oral, intravenous, intramuscular, or subcutaneous routes. The dosage regimen of this dietary therapy may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
The nucleotides may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet. Since there is no disagreeable taste to nucleotides, they could be supplied in a powdered form to be mixed with food by the patient. For oral therapeutic administration, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain sufficient active compound so as to administer at least 0.00034 g/kg body weight active compound per day. The percentage of the compositions and preparations may, of course, be varied according to the specifics of a therapeutic situation. The amount of active compounds in such therapeutically useful compositions should be such that a suitable dosage will be obtained when a compositions is administered in a suitable way.
The tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compounds sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations.
The nucleotides may also be administered parenterally or intraperitoneally. Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Finally, the nucleotides could be supplied topically with a gel, powder, salve, or patch.
As used herein, xe2x80x9cpharmacologically acceptable carrierxe2x80x9d includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated.
Of course, the nucleotide active compounds of the present invention may be administered by any of the other numerous techniques known to those of skill in the art. (For a reference on these techniques see Remington""s Pharmaceutical Science 18th Edition, (1990)) which is specifically incorporated herein in pertinent part for this purpose). Supplementary active ingredients can also be incorporated into the compositions.
The present invention also is proposed to provide a dietary supplement for the promotion of wound healing. This regimen may comprise a concentration of nucleotides therapeutically effective to promote wound healing in a pharmacologically acceptable carrier solution. The dietary supplement prepared in any suitable form, for example as a liquid suitable for injection, parenteral administration, or oral administration or as powder suitable for mixing with food or a beverage, e.g., as tablets or capsules. It is contemplated that the dietary regimen of the invention may be administered to an animal either as a pretreatment in anticipation of surgery or after a wound has occurred to both hasten and enhance the quality of wound healing.
The present invention also includes methods for promoting wound healing in animals. These methods comprise preparing a composition of nucleotides effective to promote wound healing and treating an animal with an effective concentration of the composition. These methods can be of use in treating a wound that presently exists or a wound that may exist in the future, for example, in the case of a scheduled surgery. Thus, the present formulations may be used as part of a pretreatment plan that would provide a heightened level of nucleotides in the animal prior to surgery that will in turn enhance both the healing process and the rate at which the wound is healed.
It is projected that it will be beneficial to place many, if not all, surgery patients on a nucleotide pre-treatment regimen to promote the more rapid healing of incisions, etc., that occur during surgery. For example, a patient would be given a nucleotide concentration effective to promote wound healing in any of the previously suggested forms from the time of the diagnosis of the need of surgery until a prescribed time post-surgery when the wound has healed satisfactorily. It is projected that nucleotide treatment can be done for an appropriate period prior to surgery. This period may be quite short in a stressed person, but could be as long as a number of weeks.
The present invention also contemplates methods of enhancing the rate of wound healing with the administration of a therapeutically effective concentration of nucleotides to a wounded animal. Such an enhancement will most times also involve an increase in the collagen content of a wounded area.
The present invention contemplates methods encompassing a pretreatment regimen for enhancing the rate of wound healing in an animal that is to undergo surgery. These methods comprise the administration of a therapeutically effective concentration of nucleotides in a pharmacologically acceptable carrier to an animal. A most preferred embodiment the pretreatment method is expected to involve pretreatment for up to around 4 weeks prior to surgery. However, benefits of this method can be expected with shorter lengths of pretreatment. It is anticipated that the preferred embodiments of these pretreatment methods will comprise as active compounds RNA, adenine, uracil or a mixture thereof as the source of nucleotides. Of course, those of skill in the art will understand that other sources of nucleotides will be useful as active compounds in this invention.
The present invention also contemplates a method of preparing a therapeutic agent for the promotion of wound healing comprising placing a wound healing promoting concentration of nucleotides in a pharmacologically acceptable carrier solution. The carrier should provide an adequate means for delivering the nucleotides to an animal in need thereof. This preparation could be in solid form (such as in powdered capsule or tablet form) or in a liquid form (suitable for injection, parenteral or oral administration).
The present invention therefore provides improved therapeutic agents for wound healing, methods for the preparation of these therapeutic agents, and methods for the promotion and enhancement of the rate of wound healing. These compositions and methods are anticipated to provide for a more rapid and complete wound healing in animals. As wound healing is the most catastrophic and costly problem associated with surgery, the advantages of reduced medical complications associated with the healing process and improved quality of wound healing will provide a significant advancement in patient post-surgical clinical management. These and other advantages of the present invention will be further appreciated from the detailed description provided below.
One embodiment of the present invention involves a method for promoting wound healing in a diabetic subject. This involves preparing a dietary composition supplemented with RNA, adenine, uracil, inosine or adenosine in an amount effective for promoting wound healing and feeding the diabetic subject the composition. Another important component of the present invention is a method for promoting wound healing in an animal subject to protein-deficient nutrition. This involves preparing a dietary composition supplemented with RNA, adenine, uracil, inosine or adenosine in an amount effective for promoting wound healing and feeding the protein-deficient animal with the composition. This method is further enhanced if the protein or amino acid source is also fed to the animal at this time.
In one important aspect, the present invention may involve preparing a sterile composition comprising, RNA, adenine, uracil, inosine or adenosine in an amount effective to promote wound healing and topically treating the wound of an animal with the composition. Another important embodiment of the present invention for the promotion of wound healing comprises obtaining a crosslinked collagen mesh. This embodiment further involves obtaining a composition supplemented with RNA, adenine, uracil, inosine or adenosine in an amount effective for promoting wound healing then emplacing the crosslinked collagen mesh on the wound and treating the animal with the composition. The treatment may be topical or enteral, in any manner such that the wound area has increased nucleotide concentrations. The compositions of the present invention may be designed to be intravascularly administered. For example, when a patient is on a parenteral feed, it might be expected that the feed was deficient in amino acids. Thus a parenteral feed comprising the nucleotide compositions described above as well as additional amino acids or proteins may be substituted. Topical administration of the nucleotides and possibly protein or amino acids should also be effective.